Some pathogenic Gram negative bacteria produce chemical moieties known as bacterial autoinducers (BAIs). BAIs are produced by Gram negative bacteria as a mechanism for communicating with other bacteria-when they have grown to a high cell density. This mechanism is known as quorum sensing.
BAIs assist in the transcriptional control of genes involved in a wide range of metabolic activities. When the bacterial population reaches a critical threshold, the concentration of BAIs also reach a concentration sufficient to enable the BAIs to bind a group of transcription factors, known as R-proteins. Binding of the BAIs to the R-proteins triggers binding of the newly formed BAI/R-protein complex to DNA, which then induces transcription of a group of genes. In the gram negative pathogenic bacteria, a subgroup of the activated genes are pathogenic determinants.
BAIs are small, non-immunogenic, lipid-soluble molecules which are capable of diffusing out of the bacteria, and into the environment where they enter host cells. BAIs share structural characteristics, in particular, they have a homoserine lactone ring with an N-acyl side chain. Variability between different BAIs resides primarily in the structure of the acyl side chain.
It has been proposed that in addition to regulating transcription in bacteria, the BAIs also regulate transcription in cells of an infected mammalian host. A list of presently known bacterial autoinducers (BAIs) and the Gram negative bacteria which produce them are identified in Table 1 below:
TABLE 1Gram negative bacteria:Bacterial autoinducer (BAI):Aeromonas hydrophilaAHAIAgrobacterium tumefaciensN-(3-oxo)-octanoyl-L-homoserine lactone(OOHL)Burkholderia cepaciaN-octanoylhomoserine lactoneChromobacterium violaceumN-hexanoyl-L-homoserine lactone (HHL)Enterobacter agglomeransN-(3-oxo)-hexanoyl-L-homoserine lactone(OHHL)Erwinia stewartiOHHLErwinia carotovoraOHHLEscherichia coliStructure not yet determinedNitrosomas europeaOHHLPhotobacterium fischeriOHHL, OOHL; OHLPseudomonas aeruginosaN-(3-oxododecanoyl)-L-homoserinelactone (PAI-1);N-(butanoyl)-L-homoserine lactone (PAI-2)Pseudomonas aureofaciensStructure not yet determinedRhizobium leguminosarumN-(3-hydroxy)-tetradecanoyl-L-homoserine lactone (HtDeHL)Serratia liquefaciensPAI-2Vibrio fischeriOHHLVibrio harveyiN-(3-hydroxy)-butanoyl-L-homoserinelactone (HBHL)Yersinia enterocoliticaOHHL, HHL
The Gram negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in immunocompromised hosts. PAI-1 has been shown to inhibit the proliferation of lymphocytes in vivo and downregulates expression of tumor necrosis factor and interleukin-12 (Telford et al., 1998, Infect Immun. 66(1):36-42). Pseudomonas aeruginosa frequently colonizes the lungs of individuals with cystic fibrosis (Hoiby, N., 1974, Acta Pathologica Microbiolo. Scand. Sect. B. 82: 551-558; Reynolds et al., 1975, Ann. Intern. Med. 82:819-832). This bacterium produces a number of extracellular virulence factors including exotoxin A, which is encoded by the toxA gene (Iglewski, B. H. and Kabat, D., 1975, Proc. Natl. Acad. Sci. USA. 72:2284-2288; Iglewski et al., 1978, Proc. Natl. Acad. Sci. USA. 75:3211-3215); an elastolytic protease encoded by the lasA gene; an elastolytic protease encoded by the lasB gene; and an alkaline protease encoded by the aprA gene (Morihara, K. and Homma, J. Y., 1985, Bacterial Enzymes and Virulence, ed. Holder, I. A. (CRC Press, Boca Raton, Fla.) pp. 41-79; Bever, R. A. and Iglewski, B. H., 1988, J. Bacteriol. 170:4309-4313; Kessler, E. and Saffrin, M., 1988, J. Bacteriol. 170:5241-5247).
Pseudomonas aeruginosa utilizes a partially redundant quorum sensing mechanism which includes two autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) and. N-(butanoyl)-L-homoserine lactone (PAI-2) (see Table 1). These autoinducers control expression of a number of virulence factors, including the elastolytic proteases lasA and lasB, autoinducer-synthase, alkaline protease, exotoxin A and rhamnolipid synthase. (Garnbello, et al., 1993, Infection & Immunity 61:1180-84; Latifi, et al., 1996, Mol. Microbiol. 21:1137-46; Passador, et al., 1993, Science 260:1127-30; Pesci, et al., 1997, J. Bact. 179:3127-32; Seed, et. al., 1995, J. Bact. 177:654-59; and Toder, et. al., 1994, Infection & Immunity 62:1320-27.) It is the production of these virulence factors which enable Pseudomonas aeruginosa to invade and induce disease in humans.
Current treatments for Gram negative bacterial infections typically target surface antigens of the bacteria to make antibodies. Development of vaccines and diagnostic antibodies to autoinducers are hindered by the fact that autoinducers are not only non-immunogenic, but are also freely diffusible through the lipid bilayer and are not covalently attached to the bacteria Several studies have demonstrated that a non-immunogenic bacterial capsular polysaccharide may be conjugated to an immunogenic compound to generate antibodies to the capsular polysaccharide (Anderson, U.S. Pat. No. 4,673,574 (conjugation of a fragment of a bacterial capsular polymer to a diphtheria or tetanus toxin or toxoid); Wessels et al., 1990, J. Clin. Invest. 86:1428-1433 (conjugation of a polysaccharide of type III group B Streptococcus to tetanus toxoid); and Schneerson et al., 1980, J. Exp. Med. 152:361-376 (conjugation of H. influenzae type b capsular polysaccharide to tetanus toxoid and other carriers.)) However, in contrast to autoinducers which are lipid diffusible, these anti-polysaccharide treatments are designed for production of antibodies specific to surface antigens covalently attached to the bacteria, resulting in lysis of the bacteria.
While synthetic autoinducer analogs limit bacterial growth in vitro, this approach fails to harness the capabilities of an active immune response that is a potentially long-lasting and effective therapeutic or prophylactic treatment (Pearson et al., U.S. Pat. No. 5,591,872). Furthermore, although autoinducer molecules themselves can be used in diagnostic bioassays, including bioluminescence, antibiotic production, or bacterial growth, these diagnostic assays fail to provide a prophylactic or therapeutic benefit to individuals exposed to autoinducer-producing Gram negative bacteria (Bycroft et al., U.S. Pat. No. 5,593,827).